Characterization of Engineered Tissue Development Under Biaxial Stretch Using Nonlinear Optical Microscopy

Little is known about the precise mechanical stimuli that cells sense and respond to as they maintain or refashion the extracellular matrix in multiaxially loaded native or bioengineered tissues. Such information would benefit many areas of research involving soft tissues, including tissue morphogenesis, wound healing, and tissue engineering. A custom tissue culture device has been constructed that can impart well-defined biaxial stretches on cruciform-shaped, fibroblast-seeded collagen gels and be mounted on the stage of a nonlinear optical microscopy (NLOM) system for microscopic characterization of matrix organization. The cruciform geometry permitted direct comparison of matrix (re-)organization within regions of the collagen gel exposed to either uniaxial or biaxial boundary conditions and examination by NLOM for up to 6 days. In addition, sequential NLOM measurements of collagen fiber orientations within gels while stretched, unloaded, or decellularized delineated contributions of applied stretches, cell-mediated tractions, and matrix remodeling on the measured distributions. The integration of intravital NLOM with novel bioreactors enables visualization of dynamic tissue properties in culture.